Immunogenicity of a vaccinia virus-based severe acute respiratory syndrome coronavirus 2 vaccine candidate

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines provide essential tools for the control of the COVID-19 pandemic. A number of technologies have been employed to develop SARS-CoV-2 vaccines, including the inactivated SARS-CoV-2 particles, mRNA to express viral spike protein, recombinant spike proteins, and viral vectors. Here, we report the use of the vaccinia virus Tiantan strain as a vector to express the SARS-CoV-2 spike protein. When it was used to inoculate mice, robust SARS-CoV-2 spike protein-specific antibody response and T-cell response were detected. Sera from the vaccinated mice showed strong neutralizing activity against the ancestral Wuhan SARS-CoV-2, the variants of concern (VOCs) B.1.351, B.1.617.2, and the emerging B.1.1.529 (omicron). This finding supports the possibility of developing a new type of SARS-CoV-2 vaccine using the vaccinia virus vector.

At present, the widely used COVID-19 vaccines include mRNA vaccines (mRNA-1273 and BNT162b2), inactivated virus vaccines (CoronaVac, BBIBP-CorV, and COVAXIN), and adenoviral vector vaccines expressing SARS-CoV-2 spike protein (ChAdOx1-S (Vaxzevria), ChAdOx1-S (COVISHIELD), and Ad26.COV2-S) (6-8, 13-15, 26). These vaccines have already played important roles in preventing SARS-CoV-2 infection, decreasing the severity and the fatality of COVID-19. Despite this, challenges still exist in the development and implementation of COVID-19 vaccines. One challenge is the constant emergence of SARS-CoV-2 variants of concern (VOCs). VOCs alpha (B.1.1.7), beta (B.1.351), gamma (P.1), delta (B.1.617.2), and the emerging omicron (B.1.1.529) (27) often exhibit higher transmissibility and greater pathogenicity and are less responsive to the current COVID-19 vaccines, which were designed on the basis of the ancestral SARS-CoV-2 strain. Preclinical studies also showed less protection by these vaccines in the upper respiratory tract than in the lower respiratory tract (12,15,22). It is pivotal to develop COVID-19 vaccines that are able to protect against different VOCs and elicit persistent protective immunity. The combination of different vaccines, the inclusion of multiple SARS-CoV-2 antigens, and the exploration of new vaccine technologies are expected to provide solutions to these challenges.
In the present study, we aimed to engineer a VACV Tiantan strain expressing SARS-CoV-2 spike protein and evaluate its effectiveness as a new SARS-CoV-2 vaccine candidate through stimulating both humoral and cellular immune responses. The mice immunized with VACV Tiantan vector-based vaccine produced high levels of SARS-CoV-2 spike protein-specific antibodies, neutralizing antibodies (NAbs), and T-cell responses, which supports the potential use of this VACV recombinant as a SARS-CoV-2 vaccine.

Generation of vaccinia virus recombinants expressing SARS-CoV-2 spike or receptor-binding domain
To generate and test the VACV Tiantan strain-based COVID-19 vaccine candidate, we performed homologous recombination to insert SARS-CoV-2 spike protein (with the last 19 amino acids deleted) or the RBD sequence into the thymidine kinase (TK) locus in the VACV Tiantan strain under the control of the early/late promoter p7.5 ( Figure 1A). Recombinant viruses were selected by PCR using primers targeting spike protein and RBD ( Figure 1B) and further purified by five rounds of plaque selection. DNA was extracted from the purified recombinant viruses, amplified by PCR, and verified by sequencing. Primers recognizing spike protein and RBD sequences or sequences surrounding the TK region were designed for PCR ( Figure 1B). PCR analysis and further DNA sequencing confirmed the insertion of SARS-CoV-2 spike protein or RBD sequence in the genome of the recombinant viruses ( Figure 1C).

Expression of receptor-binding domain and spike protein in cells infected with vaccinia virus recombinants
Vero cells were infected with VACV-expressing spike protein (dTK-S) or VACV-expressing RBD (dTK-RBD) for 24 h. The expression of spike protein and RBD was shown by Western blotting (Figure 2A). The full-length S (~190 kDa) and the cleaved S1 (~100 kDa) and S2 (~90 kDa) were observed in the Western blotting, confirming the successful processing of spike protein (Figure 2A). RBD or spike protein expression in infected cells was also confirmed by the results of flow cytometry with the anti-RBD antibody staining ( Figure 2B). Results of immunofluorescence staining showed the cytoplasmic localization of RBD and spike protein in the infected cells. Cell surface expression of spike protein was detected in cells that were not permeabilized before immunostaining ( Figure 2C). In agreement with the report that SARS-CoV-2 spike protein induces membrane fusion of ACE2-expressing cells, syncytia were observed for dTK-S infected HeLa-ACE2 cells but absent in the control dTK-or dTK-RBD-infected cells ( Figure 2D). These data demonstrate that the engineered VACV expresses SARS-CoV-2 spike protein at the cell surface, which is fusogenic.

Vaccinia virus dTK-S and dTK-RBD elicit high titers of spike protein-specific antibodies and T-cell responses in mice
To evaluate the antigenicity of VACV recombinants, BALB/ c mice were immunized with VACV control dTK, vaccine candidates dTK-S, or dTK-RBD at doses of 10 5 plaqueforming unit (PFU) (low dose) or 10 6 PFU (high dose) by intramuscular route at week 0 and week 3 ( Figure 3A). Spike protein-or RBD-binding antibodies in the sera at week 2 and week 5 were measured by ELISA ( Figures 3B, C). The levels of spike protein/RBD-specific antibodies were higher in dTK-S-vaccinated mice than those in dTK-RBD-vaccinated mice. The high dose of 10 6 PFU VACV immunization produced higher levels of antibodies. The dTK-RBD vaccination group exhibited no S1-binding antibody and moderate RBD-binding antibody. Levels of spike protein antibodies increased by one log 2 weeks after boosting in the dTK-S-vaccinated group. Isotype analysis of the S1-binding antibody indicated a dominant T helper 1 (Th1) cell polarization of the immune response in the dTK-S vaccination group ( Figure 3D). To assess SARS-CoV-2 specific cellular immunity, splenocytes from dTK or dTK-S (high dose) vaccination groups were obtained and stimulated with S peptides. S-specific CD8 and CD4 T cells producing IFNg, TNFa, IL-4, and IL-10 were measured using an intracellular cytokine staining assay. Upon S-specific stimulation, splenocytes from mice vaccinated with dTK-S produced greater numbers of IFNg+ CD8 and IFNg+ CD4 T cells as well as TNFa+ CD8 and TNFa+ CD4 T cells than did the control dTK group and induced a Th1-biased immune response ( Figure 3E).
Although the neutralizing activity against B.1.1.529 decreased, all of the five mice elicited positive NAb response against the omicron variant ( Figure 4G). Together, these data demonstrate that the dTK-S VACV elicits a strong NAb response to the ancestral Wuhan SARS-CoV-2 and VOCs B. been tested using a highly attenuated VACV strain Modified Vaccinia Ankara (MVA) and exhibited protection in immunized animals (22)(23)(24)(25)(44)(45)(46)(47)(48)(49). Recombinant MVA-expressing spike protein or RBD were shown to elicit strong NAb responses, produce S-specific CD8 T cells, and protect vaccinated animals against a lethal intranasal SARS-CoV-2 challenge. The VACV Tiantan strain was widely used as a smallpox vaccine in China and has been successfully adopted as a viral vector to develop vaccines against a variety of virus infections, such as influenza virus, Ebola virus, SARS-CoV, HBV, HCV, and HIV-1 (35)(36)(37)(38)(39)(40)(41). The VACV-based HIV-1 vaccine candidate has completed a phase IIa clinical trial (NCT01705223). Here we showed that the VACV Tiantan strain-based SARS-CoV-2 vaccine candidate (dTK-S) induced high levels of spike protein-specific antibodies in mice. Sera from the immunized mice exhibited neutralization activity against the ancestral Wuhan stain and VOCs including beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529). The dTK-S virus also induced a predominantly Th1-type response to spike protein. These data support the potential of further developing dTK-S into a new SARS-CoV-2 vaccine.
Intranasal administration of vaccines based on the VACV was reported to stimulate both efficacious mucosal and systemic immune responses. For example, Xie et al. generated a recombinant VACV harboring Zaire Ebola virus glycoprotein (GP) that, when administered in mice via the intranasal route, induced GP-specific IgA and IgG in serum, nasal wash, and vaginal wash, as well as specific cellular immunity (37). Similarly, intranasal delivery of VACV vaccine candidates targeting the SARS-CoV-2 induced spike protein-specific IgG and IgA in serum (47,50). The increased mucosal immune response supports the high potential of recombinant VACV to prevent mucosal infection of SARS-CoV-2. It is difficult to compare the effectiveness of vaccine candidates based on the VACV Tiantan strain and those derived from other VACV strains because these VACV vaccine candidates have been evaluated with different immunization routes including intravenous injection, dTK-S VACV elicits effective humoral and cellular immunity in mice. (A) Immunization scheme. BALB/c mice aged 6-8 week were intramuscularly immunized with 1 × 10 5 or 1 × 10 6 PFU of control dTK, dTK-S, or dTK-RBD at week 0 and week 3. Sera were collected 2 weeks after each vaccination for further quantification of antibody responses. (B-D) Humoral responses in sera of immunized mice were evaluated. S1-binding antibody (B) and RBD-binding antibody (C) were analyzed by ELISA. The IgG2a/IgG1 ratio of S1-binding antibody in dTK-S immunized mice was detected by ELISA using HRP-conjugated isotype-specific antibodies (D). (E) T-cell response induced by dTK-S. CD8 and CD4 T cells expressing IFNg, TNFa, IL-4, and IL-10 in the spleen of dTK-or dTK-S-immunized mice (high dose) were evaluated by intracellular cytokine staining, after stimulation with S peptides. Dotted lines indicate the lower limit of detection (LLOD). Animals with a response at or below the LLOD were put on LLOD. Statistical differences as determined by Student's t-tests are indicated by asterisks; *p < 0.05, **p < 0.01; ***p < 0.001. 1st, sera collected 2 weeks after prime; 2nd, sera collected 2 weeks after boost; L, low dose (10 5 PFU); H, high dose (10 6 PFU); N, dTK immunization; S, dTK-S immunization; RBD, dTK-RBD immunization. dTK-S, vaccinia virus-expressing spike protein; VACV, vaccinia virus; PFU, plaque-forming unit; RBD, receptor-binding domain; HRP, horseradish peroxidase. intramuscular injection, intranasal injection, intraperitoneal injection, and different vaccination strategies such as prime only or prime-boost, and low or high injection doses (22, 24, 47, 51). Further research is warranted to characterize in detail the impact of different immunization routes and vaccination strategies on the effectiveness of VACV Tiantan candidates. It was reported that VACV-induced antibodies remained stable for 1 to 75 years after vaccination, and VACV-specific cellular responses declined with a half-life of 8 to 15 years (52).
The level of RBD-specific IgG in RBD-expressing VACV immunized mice sustained for 195 days post-immunization (53), further supporting the long-lasting immune response elicited by VACV-based vaccines.
We found that the dTK-RBD did not induce as strong humoral immunity as the dTK-S despite the high expression of RBD in the infected cells. dTK-S may present more epitopes for antibody production, or membrane-associated spike protein exhibits stronger immunogenicity than soluble RBD. . Animals with a response at or below the LLOD were put on LLOD. Statistical differences as determined by Student's t-tests are indicated by asterisks; *p < 0.05, **p < 0.01; ***p < 0.001. dTK-S, vaccinia virus-expressing spike protein; VLP, virus-like particle.
Membrane-anchored immunogens have been shown to elicit greater immune responses than cytosolic expressions in an MVA vector (54,55), and recombinant MVA RBD encoding a membrane-bound RBD showed higher immunogenicity than soluble RBD (25). Recombinant VACV Tiantan expressing the membrane-bound RBD may increase its immunogenicity, but further studies are needed to test this hypothesis.
In conclusion, we have generated a SARS-CoV-2 vaccine candidate using the VACV Tiantan strain. Mice immunized with dTK-S mount robust humoral immune responses to spike protein of both the ancestral SARS-CoV-2 and the circulating VOCs. Meanwhile, this vaccine candidate also induced S-specific T-cell responses, thus holding promise as another tool to be added to the COVID-19 vaccine arsenal.

Construction and purification of vaccinia virus recombinants
Codon-optimized cDNA clones encoding SARS-CoV-2 spike (with C-terminal 19 amino acids deletion) or the RBD were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). Spike protein and RBD sequences were inserted into the thymidine kinase (TK) locus of the VACV Tiantan strain under the transcriptional control of the early/late promoter p7.5 by homologous recombination. After five rounds of plaque purification, insertion of spike or RBD into the viral genome was confirmed by PCR and DNA sequencing. Purified recombinant viruses were amplified by infecting Vero cells and titrated by plaque assay. Primers used were as follows: J1RJ3R-F, CAGATTTCTCCGTGATAGGT; J1RJ3R-R, CTTAGTA AATCCGCCGTACT; S-F, GGATCCATGTTCGTTTTC CTTG; RBD-F, CGCGTCCAGCCAACC; RBD-R, CTTCTTG GGACCGCATAC; TK-F, GAAGGACAGTTCTTTCCAG ACATTGTTG.

Western blotting
Vero cells were infected with VACV recombinants expressing SARS-CoV-2 spike protein or RBD. Cell lysates were harvested 24 h post-infection; samples were boiled for 10 min, separated in a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel, and transferred onto nitrocellulose filter membranes. After being blocked with 5% milk, membranes were probed with the antibodies anti-SARS- Flow cytometry dTK-S-or dTK-RBD-infected Vero cells were permeabilized with 0.1% Triton X-100 and incubated with the anti-RBD antibody for 1 h, followed by incubation with Alexa Fluor 488-conjugated donkey anti-rabbit antibody (Thermo Fisher Scientific, Waltham, MA, USA; A11034). Samples were analyzed with FACS Canto II (BD Biosciences, San Jose, CA, USA).

Immunofluorescence assay
HeLa cells were infected with VACV recombinants expressing SARS-CoV-2 spike protein or RBD for 24 h and fixed with 4% paraformaldehyde for 10 min. Cells were permeabilized with 0.2% Triton X-100 for 10 min. Permeabilized and un-permeabilized cells were blocked with 5% bovine serum albumin (BSA) and then incubated with the anti-RBD antibody for 1 h, followed by incubation with Alexa

Immunization of animals
All animal experiments were performed in accordance with guidelines and regulations and approved by the Institutional Animal Care and Use Committee of Beijing Protein Innovation Co., Ltd. Female BALB/c mice aged 6 to 8 weeks were intramuscularly immunized with 1 × 10 5 and 1 × 10 6 PFU of dTK, dTK-S, or dTK-RBD at week 0 and week 3, respectively. All animals were euthanized following the animal use protocols 2 weeks after the last immunization. Sera were collected 2 weeks after each vaccination for further quantification of antibody responses.
Detection of S1/RBD-binding antibodies by ELISA S1-binding antibodies were detected with SARS-CoV-2 Spike S1 Antibody Titer Assay Kit (Mouse) (Sino Biological Inc., KIT007). RBD-binding antibodies were detected with Spike RBD Antibody Titer Assay Kit (Mouse) (Sino Biological Inc., KIT006) according to the instructions of the manufacturers. Briefly, sera were serially 4-fold diluted in dilution buffer, and 100 ml of the diluted sera was added to spike S1/RBD precoated microplates and incubated for 2 h at room temperature. After the microplates were washed, diluted horseradish peroxidase (HRP)-Rabbit anti-Mouse IgG was added to the plates and incubated for 1 h at room temperature. The plates were then incubated with substrate solution for 20 min. Levels of antibodies were determined by measuring the optical density (OD) at 450 nm with a microplate reader after termination with stop solution. Final end point titers (1/n) were defined as the highest dilution that yielded an absorbance >0.1. For detection of antibody isotypes, HRP-conjugated Goat anti-Mouse IgG1 (Abcam, Cambridge, UK; ab97240) and HRP-conjugated Goat anti-Mouse IgG2a (Abcam, ab97245) were used.

Pseudovirus neutralization assay
Pseudoviruses were produced by co-transfection 293T cells with psPAX2 encoding the structural polyproteins Gag-Pol, pLenti-GFP containing the GFP and luciferase reporters, and cDNAs encoding SARS-CoV-2 spike protein (last 19aa deleted) or the indicated mutants. Supernatants were harvested at 48 h post-transfection and passed through 0.45-mm filters. Twofold serial dilutions of the heat-inactivated sera were mixed with the above pseudoviruses for 1 h at 37°C and then added to HeLa-ACE2 cells in a 96-well plate. The luciferase activities were determined using the Steady-Glo Luciferase Assay System (Promega, Madison, WI, USA; E2520) 48 h post-infection. The neutralization titers were defined as the reciprocal of the highest dilution at which a 50% reduction was observed relative to the uninfected control wells.

rVSV-eGFP-SARS-CoV-2 neutralization assay
Replication-competent recombinant VSV-SARS-CoV-2 viruses (rVSV-eGFP-SARS-CoV-2) in which VSV-G was substituted with the SARS-CoV-2 S protein were gifts from Dr. Aihua Zheng (42). Twofold serial dilutions of the heat-inactivated sera were mixed with rVSV-eGFP-SARS-CoV-2 at 37°C and then added to 293T-ACE2 cells in a 96-well plate. The GFP-positive cells were calculated by flow cytometry 24 h postinfection. The neutralization titers were determined as the highest dilution at which a 50% reduction was observed.

VLP neutralization assay
SARS-CoV-2 VLPs were produced by co-transfecting 293T cells with CoV2-N, CoV2-M-IRES-E, CoV-2-Spike, and Luc-PS9, which were gifts from Jennifer Doudna (Addgene, Cambridge, MA, USA; plasmid # 177950, 177938, 177939, and 177942) (43). Supernatants were harvested at 48 h post-transfection and passed through 0.45-mm filters. Twofold serial dilutions of the heatinactivated sera were mixed with the above VLPs for 1 h at 37°C and then added to 293T-ACE2 cells in a 96-well plate. The luciferase activities were determined using the Steady-Glo Luciferase Assay System (Promega, E2520) 24 h post-infection. The neutralization titers were determined as the highest dilution at which a 50% reduction was observed relative to the uninfected control wells.

SARS-CoV-2 neutralization assay
Twofold serial dilution of the heat-inactivated sera was mixed with 100 TCID 50 SARS-CoV-2 (WH-09, GenBank: MT093631) for 1 h at 37°C in a 5% CO 2 incubator and then added to Vero cells in 96-well plates. The plates were incubated for 3-5 days to observe cytopathic effect (CPE). The serum dilution at which 50% of the cells were protected against infection was calculated.

Data availability statement
The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding authors.

Ethics statement
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee of Beijing Protein Innovation Co., Ltd.

Author contributions
FG, FX, FZ, and BA conceived the project. FX, SM, FZ, XL, ZF, YH, LWe, YMH, YX, and LWa performed the experiments. CL, FX, and FG wrote the manuscript. All authors reviewed the manuscript and discussed the work. All authors contributed to the article and approved the submitted version.


Tumor Microenvironment in Acute Myeloid Leukemia: Adjusting Niches

Acute myeloid leukemias (AML) comprise a wide array of different entities, which have in common a rapid expansion of myeloid blast cells leading to displacement of normal hematopoietic cells and also disruption of the microenvironment in the bone marrow niches. Based on an insight into the complex cellular interactions in the bone marrow niches in non-neoplastic conditions in general, this review delineates the complex relationship between leukemic cells and reactive cells of the tumor microenvironment (TME) in AML. A special focus is directed on niche cells and various T-cell subsets as these also provide a potential therapeutic rationale considering e.g. immunomodulation. The TME of AML on the one hand plays a vital role for sustaining and promoting leukemogenesis but - on the other hand - it also has adverse effects on abnormal blasts developing into overt leukemia hindering their proliferation and potentially removing such cells. Thus, leukemic cells need to and develop strategies in order to manipulate the TME. Interference with those strategies might be of particular therapeutic potential since mechanisms of resistance related to tumor cell plasticity do not apply to it.

INTRODUCTION
Acute myeloid leukemia (AML) can be defined as a clonal expansion of hematopoietic progenitor cells showing a stop of differentiation and maturation at various stages (1). AML has a severe impact on affected patients with a general five-year survival rate of 24%, a median survival of 8.5 months, and an even worse prognosis in the most commonly affected patient group of people over the age of 65 showing a median survival of only 2.7 months (2). This data and the fact that no increase in survival rates could be achieved in the last decades, show the urgent need for improved treatment modalities.
The mainstay of AML therapy includes intensive chemotherapy and allogeneic hematopoietic cell transplantation (AHCT) (3). The rationale behind AHCT is the idea that blasts remaining after chemotherapy will be eliminated by the transplanted donor-immune cells (graft-versus-leukemia effect). However, in a considerable fraction of patients, relapses occur. On the other hand, manyespecially elderly -AML patients are not eligible for this therapeutic option (4) and, thus, other therapeutic strategies must be explored. Improved understanding of the biology of AML and especially its microenvironment, including the exploration of the applicability of chimeric antigenreceptor T cells (CAR-T), might open new treatment options and significantly improve the outcome of patients. A key to achieve this goal is a profound understanding of the bone marrow niche, which provides physical protection and release of pro-survival factors for leukemia cells (5,6), and the interaction between AML blasts and the immune system as potential target for immunotherapeutic approaches (7,8).
As hematopathologists, we are confronted daily with evaluation of bone marrow biopsies and the characterization of its cellular components. This work has resulted in the conceptualization of various own studies dealing with the TME of hematopoietic neoplasms, which will be presented in this review along with seminal publications of other groups. So the focus of this review is the TME of myeloid tumors, particularly AML, from the viewpoint of histopathology that may mechanistically explain how AML blasts interact with the TME to support growth and survival. For a detailed view on therapeutic approaches targeting the TME in AML, we refer to other excellent reviews focusing on that topic (9, 10).

THE MICROENVIRONMENT OF THE BONE MARROW NICHE
The term "bone marrow niche(s)" has been coined to describe the specialized interplay of various cells and soluble factors providing the basis for sufficient and well-regulated hematopoiesis from the hematopoietic stem cells (HSC).
The bone marrow consists of a delicate vascular architecture of arterioles, veins and specialized sinusoids that enable trafficking of cells and soluble factors to the blood stream and vice versa, particularly through the fenestrated basal lamina of the sinusoids (11). Another important component of the bone marrow niches are stromal cells, which to a large extent comprise the adipose tissue, a longtime neglected component (12). Many authors distinguish a perivascular and an endosteal niche (10). It is still a matter of debate whether this distinction really can be made, since there is cumulating evidence in the support of the existence only of the former (13).
Hematopoiesis occurs in a circadian fashion effectuated by sympathetic nerve fibers in the bone marrow (14), and HSC themselves have been shown to be subject to respective oscillation, which is orchestrated by the sympathetic nervous system (15). Signaling via b3-adrenergic receptors regulates nestin-expressing mesenchymal cells and their cell progenitors (MSC) (16), which are of core importance for the perivascular bone marrow niches (17). As a net effect, b3-adrenergic signaling e.g. regulates trafficking of HSC (18).
The endosteal bone marrow niche (17) has been described as a shelter for HSC, and its regulation is orchestrated by osteoblasts and their progenitors (19). Osteoblasts form protective layers for HSC, they can also keep them in a non-circulatory state via cellcell adhesion molecules (20). This niche is preserved after treatment by chemotherapy and radiation, being the source of bone marrow renewal after such insults (21). As also capillaries seem to play an as important role in the endosteal niche as in the perivascular niche, it has been proposed to relinquish the separation between different niches. Just recently Panvini et al. presented human data on the presence of nestin+ capillary-like tubes (NCLTs), not surrounded by sub-endothelial perivascular cells in direct contact to the bone line and spatially correlated with hematopoietic stem/progenitor cells and possibly involved in regulating human hematopoiesis within the endosteal compartment (22). This group also showed that the endosteal niche capillary network gets destroyed in the course of AML evolvement in favor of the central perivascular niche.
The perivascular niche is considerably (about 9 times) larger. Its MSC can differentiate into osteoblasts, adipocytes, chondrocytes and fibroblasts (23), and are particularly important for HSC by secreting factors such as C-X-C motif chemokine 12 (CXCL12), stem cell factor (SCF) and interleukin (IL) 7 (16). Vice versa, MSC can be modulated by HSC to improve their own microenvironment (24). The most voluminous compound of the perivascular niche, the adipocytes of the bone marrow, have been shown to be different from adipocytes elsewhere in the body. Their amount steadily increases during lifetime and they are involved in HSC maintenance and proliferation by secreting adipokine and adiponectin (25), in addition of being an important source of nutrients for bone marrow cells. Production of CXCL12, IL3 and IL6 has also been documented for this cell type (26). Finally, the vascular partition consists of sinusoids and arterioles, whichbased on single cell analysismust be regarded as different subcompartments (27,28), e.g. sinusoids being mainly involved in cell egression from the bone marrow, while arterioles play a central role in nutrient and oxygen supply.
Macrophagesas in many other forms of TMEalso are a pivotal part of the bone marrow niches. They can give rise to osteoclasts, influence osteoblasts and CXCL12-secretion by MSC to maintain homeostasis of HSC (29). Interestingly, the progenies of HSC, megakaryocytes also play a backloop role for HSC by secreting several cytokines such as insulin-like growth factor 1, platelet factor 4 and transforming growth factor beta (TGFb) (30).

THE TUMOR MICROENVIRONMENT OF MDN AND MPN: SIMILARITIES AND DIFFERENCES TO AML
A weighty proportion of AML cases does not present as de novo disease but represents clinical or at least genetically perceptible evolution (31) of background clonal hematopoietic stem cell disorders such as myelodysplastic syndromes (MDS) that are now proposed to be called myelodysplastic neoplasms (MDN), or myeloproliferative neoplasms (MPN), the microenvironment of which has been the focus of research. MDN patients carry a high risk (up to 25%) of developing AML (32), while in MPN this risk is more variable, depending on the respective entity, and is below 10%. Interestingly in this regard, MDN cells can induce transformation of normal MSC into MSC with proinflammatory characteristics, which can then support leukemic progression (33), while vanishing nestin-positive niches is a well-established mechanism of MPN pathogenesis (34). Moreover, it has been shown in animal models that certain molecular dysfunctions in MSC can induce an MPN-like phenotype (35,36). This just shows two brief insights into the interaction and relationship of neoplastic ells and their environment of these disease groups. In the following paragraphs when focusing on different cell subsets and soluble factors, we will figure out current knowledge in regard to the TME of AML.

THE ROLE OF MESENCHYMAL STEM CELLS AND THEIR PROGENIES IN AML
MSC are pluripotent cells that can differentiate into different types of mature cells as described above. During lifetime, there is a "physiological" change of MSC, which is reflected by increase of adipocytes and loss of osteoblasts (37). This alteration might also explain the well-known increasingly reduced bone marrow cellularity in the course of life. The underlying change of MSC differentiation is achieved via different mechanisms, including altered cytokine levels, resulting in decreased Wnt-related signaling and intensified RhoA-related signaling (38) as well as reduced Runx2 signaling, which is important for osteoblastic differentiation (39). "Senescence" that can be attributed to (cumulative effects of) pro-inflammatory cytokines, and changes of the sympathetic nerve system during life affect MSC (40). Indeed and as a cumulative result of altered signaling in cancer, nestin-positive MCS are significantly reduced in several hematologic malignancies. In a study on the TME of MPN, we could demonstrate that there is both a reduction of sympathetic nerve fibers and, consecutive, of nestin-expressing MSC in MPN, which is linked to the effects of proinflammatory cytokines that are increased in MPN, and can be to a part reverted by administering b3-adrenergic agonists (34). Interestingly, reduced numbers of nestin-positive MSC also correlate with another inflammatory condition, GvHD in AHCT patients (41). On the other hand, the role of nestin-positive MSC is different in AML as here, no depletion of these MSC has been observed ( Figures 1A, B) and it is speculated that AML blasts are in need of MSC for survival and chemotherapy resistance, being dependent on the oxidative phosphorylation and tricarboxylic acid cycle activity, and antioxidant defense of the latter (42).
Whether the genuine disruption of MSC niches in MPN (34) compared to rather increased MSC-niche densities MDN (43) may additionally explain the higher transformation rate into AML of the latter, is an intriguing hypothesis that remains to be resolved.
Analogously to what has been mentioned in the previous section discussing the relationship between MPN and their TME, mutations can also occur in the MSC themselves, which is documented in MPN linked to mutations in Ptpn11 (44) or progression of AML fostered by Ctnnb1 mutations in osteoblasts (45) in mouse models. This shows a new path of leukemogenesis, initiated not by mutations occurring in HSC but in MSC with secondary mutations occurring in the blasts. Similar to what is known from studies on bone marrow senescence, disruption of sympathetic nerve signaling also happens in AML. In mouse models on both AML and MPN, the destruction of Schwann cells and reduced sympathicotonus significantly altered bone marrow niches and promoted the increase of leukemic stem cells and disease progression (34,46). Interestingly, b3-adrenergic agonists helped to restore physiological conditions and prevented disease progression.
AML blasts can push MSC to differentiate into osteoblasts, being as supportive for leukemia as for HSC in general (47), andvia the mechanism of leukemic stem cell protection -in the endosteal niche in particular (48). This seems to occur via secretion of proinflammatory cytokines such as CCL3 or thrombopoietin (TPO). Single cell RNA sequencing studies could already provide deeper insights into the reprogramming of MSC in vivo: Baryawno et al. elegantly demonstrated that different MSC are involved, including e.g. significant changes in osteoblast subsets and decreases differentiation into adipocytes, accompanied by upregulation of hypoxia-related genes in MSC (28).
Adipocytes are getting diminished by AML expansion in the bone marrow, which seems to be not only due to spatial disproportion, but also by preferential MSC-differentiation towards the osteoblastic lineage (49). On the other hand, adipocytes provide nurture to AML cells via release of fatty acids and also are important for bioavailability/unavailability of many lipophilic drugs (50), so their importance for AML cells is still difficult to interpret. Interestingly, the latter property of these cells is suspected being one reason for the inferior survival of elderly AML-patients as adipocyte-rich niches may provide a protective environment for AML blasts (51).
AML blast tightly interact with the endothelium. It has long been known that AML show an increased microvascular density compared to non-neoplastic bone marrow (52). Vascular endothelial growth factor (VEGF) is a major player in this relationship: besides fostering angiogenesis it can also inhibit apoptotic signaling in AML cells and support proliferation by upregulating secretion of GM-CSF by endothelial cells (53). Moreover, adhesion of leukemic stem cells through E-selectin to the vascular niche protects the former from the lethal effect of chemotherapy, which can be specifically counteracted (54).
Extracellular matrix components are an important factor for cell (im-)mobility and, thus, also a vital component of the TME. Already ten years ago, we could show the importance of RHAMM/CD168 (Figure 2A), the receptor for hyaluronic acid-mediated motility, as a negative prognostic marker for AML patients (55). CD44 that also interacts with hyaluronic acid (Figure 2B), is a type I transmembrane protein serving -especially in its sialofucosylated form -as adhesion molecule with ligands such as osteopontin, E-and L-selectin and fibronectin, and is an important factor for homing of HSC, and particularly of leukemic stem cells (56). Blocking CD44, the most commonly variant of which expressed in AML is v6 that is moreover linked to poor outcome, has been shown to prevent persistence of AML blasts in the bone marrow niche upon chemotherapy, which is otherwise linked to the quiescentpromoting-and, thus, cell-protective TME functions of CD44 (57).

THE ROLE OF LYMPHOCYTES AND MONOCYTES IN AML IMMUNE ESCAPE
As seen in many other tumors, immune escape is an important survival strategy for AML. AML blasts have been shown to impede the formation of immune synapses (58) and impair cytotoxic activity of T-cells (59). Generally, and in AML in particular, immune escape can be divided into different strategies including hiding of the malignant cells from the immune system up to manipulation of various immunecell subsets.
T-cells can be divided into regulatory and cytotoxic T-cell subgroups. Regulatory T-cells (Tregs) dampen inflammatory responses, both by secreting anti-inflammatory cytokines as well as silencing cytotoxic T-cells (60). Thus, Tregs' effects to provide protection from the immune system may be important for AML. AML blasts express one of the most potent cell-contact immune silencers, programmed death ligand 1 (PD-L1; Figures 3A, B), which increases with disease progression (61). They can also produce reactive oxygen species (62) and indolamine-2, 3-dioxygenase (IDO), which both promote differentiation towards Tregs (63). Just recently, a study by Ragaini et al. demonstrated that an IDO1-related immune gene signature predicts overall survival in AML (64). Another target to induce T-cell anergy is T-cell immunoglobulin and mucin domain 3 (TIM3), which binds to and is activated by galectin-9, the latter being highly expressed on AML blasts, which leads to an activation of several downstream signaling pathways such as the MAPK/ERK, PI3K-and AKT (65). TIM3 can stimulate the production of IDO and thus foster immune evasion (66). Yet, regarding the role of TIM3-expression AML, there are still conflicting results (67)(68)(69). Secretion of inducible T-cell co-stimulator ligand (ICOSL) and IDO's product, N-formylkynurenine, furthermore contribute to an immunosuppressive environment stimulating the expansion of Tregs, while limiting cytotoxic activity (70,71). Low Treg levels have been generally associated with better outcomes in AML (72). However the very dynamic nature of these interaction can be appreciated from the following example: in a retrospective cohort we could show higher numbers of FoxP3+ Tregs ( Figure 4A) in the early phases after induction therapy are associated with higher complete remission rates and better overall AML survival (73), contrasting their role when assessed  at baseline AML biopsies (74). We hypothesized that this is attributed to active changes of the T-cell compartment reflecting the stereotypic recovery of the immune system after chemotherapy. Due to their immunomodulatory abilities, the expanding Tregs could foster the recovery of normal hematopoietic cells effectuated e.g. by limiting activity of effector T-cells, which may otherwise react to potential neoepitopes unmasked due to cell destruction caused by chemotherapy. Accordingly, the beneficiary role of Tregs becomes evident when looking at lower rates of GvHD in Treg-high AHCT patients (75). Alongside, high expression levels of the surface molecules mentioned such as PD1/PD-L1 and TIM3 are associated with worse prognosis in AML, thus suggesting potential relevance for therapeutic interventions, with first immune checkpoint inhibition trials being underway (76) althoughas alluded to above -investigations of the expression of these markers still produces conflicting results (69). Yet and in accordance with the described above, it seems that immune checkpoint inhibition as a single therapy is not successful, therefore currently combination therapies are being explored (77).
T-cell exhaustion can further be achieved by alterations of the metabolic microenvironment of AML, which can be characterized as glutamine-rich (78) and an arginine-deprived (79). Therefore, administering L-asparaginase (80) and inhibiting arginine deprival (81) have been both shown to be promising options for AML. Further immunomodulating drugs such as lenalidomide have also been tested in AML. In a translational side-project of a randomized clinical study (HOVON103 AML/SAKK 30/10), addressing the role of TME in AML treated by chemotherapy with or without addition of lenalidomide, we could show that its addition may be beneficial to (elderly) patients suffering from AML with multilineage dysplasia, where it led to a reduction of microvascularization and, likely, to an intensified specific T cell-driven anti-leukemic response (82). Lenalidomide promotes the degradation of two transcription factors, Aiolos and Ikaros, of the cereblonmediating signaling (83,84). Lenalidomide affects both neoplastic cells -by facilitating apoptosis, and the surrounding T-cells -by activating them via enhanced secretion of IL 2 (85). Indeed, under lenalidomide, the amount of T-bet+ T cells more consistently increased (82), which might be interpreted as a sign

Menter and Tzankov
Tumor Microenvironment in AML Frontiers in Immunology | www.frontiersin.org February 2022 | Volume 13 | Article 811144 of increased T cell-driven immune response against the tumor cells. Consistently, this T-bet+ T-cell subgroup ( Figure 4B) is a significant contributor to T-cell activation (86), and has a potential beneficiary effect on AML outcome as it fosters anti-AML immunity (87). The role of B-cells has not been investigated at a larger scale so far. Cheng et al. investigated the prognostic importance of the TME in AML and showed a negative impact of memory B-cells (yet not statistically significant) on survival, while increased numbers of naïve B-cells had a positive impact (88).
Besides T-cells of the adaptive immune system, AML blasts also alter the function of innate immune system cells, namely NK cells and macrophages. AML blasts downregulate surface molecules needed for their recognition by NK cells via the receptor natural killer group 2 member D (NKG2D), and release altered NKG2D-ligands reducing the cytotoxic activity of NK cells (89,90). Another mechanism to evade NK-cell recognition and, thus, destruction is inhibiting release of interferon g (91). Similar to solid tumors, AML blasts induce a shift of macrophages towards M2 polarization, M2 macrophages being immunosuppressive, supporting angiogenesis and tissue repair (92). Elevated levels of M2 macrophages in AML patients have been described in vivo and in mouse models (93). Another population manipulated by AML blasts are myeloid-derived suppressor cells, which are derived from monocytes (94). These cells cause T-cell inactivity via several mechanisms ranging from PD-L1 expression to secretion of cytokines such as IL10 and/or TGFb. In line with M2-macrophages and Tregs, these myeloid-derived suppressor cells are also more abundant in AML patients (94), and, consistently, they seem to be a risk factor for disease progression of MDN to overt AML (95). Finally, AML blasts also show a defective antigen-presentation by downregulation of human leukocyte antigens (HLAs) helping to render them invisible to immune cells (96).
These complex interactions between AML cells and nonneoplastic immunomodulating cells seems to represent a major source of difficulties for applying CAR-T in such instances (97), besides difficulties in identifying targets not also expressed by non-neoplastic HSC (98). As detailed above, induction of myeloid-derived suppressor cells and Tregs significantly reduces T-cell responses, which also holds true for CAR-T responses. Targeting CD33, which is not only expressed on AML blasts, but also on the myeloid-derived suppressor cells is a potential strategy to overcome this dilemma (99). Respecting AML-TME interactions, a phase I/II trial is currently running to explore the application of CD44v6 CAR-T in AML (https:// clinicaltrials.gov/ct2/show/NCT04097301). Constructing CAR-T with costimulatory domains to dampen the influence of Tregs might be another potential approach (100). A further obstacle to the efficacy of CAR-T is the secretion of various soluble factors by AML blasts, which will be discussed in the next paragraph.

SOLUBLE FACTORS AFFECTING THE TME
AML cells depend on numerous cytokines and soluble factors provided by cells of the bone marrow niches. In addition, several adhesion factors seem to be essential for AML cells within these niches. CXCL12 ( Figure 5A) is secreted by MSC under the orchestration of sympathetic nervous signaling, and regulates leukocyte trafficking as mentioned above (101). Interactions with its receptor, CXCR4, increase retention of HSC to the bone marrow niches and decreases their progenies' migration into the bloodstream. Several years ago, we could show that active CXCR4 signaling is associated with an inferior prognosis in AML as a consequence of an increased retention to the bone marrow associated with an enhanced chemoresistance of leukemic cells (102). Indeed, under chemotherapy, AML cells often overexpress CXCR4 ( Figure 5B) that facilitates overcoming apoptotic stimuli and improving cell survival by entering a state of quiescence and stromal protection, thus, being less amenable to the effects of cytotoxic drugs (103). CXCR4 antagonists such as plerixafor or the monoclonal antibody ulocuplumab are currently investigated in several  clinical trials (104). CXCR4-antagonists mobilize leukemic stem cells into the blood stream and block several survival pathways as discussed above, thus rendering AML cells more chemosusceptible. Importantly, granulocyte colony-stimulating factor (G-CSF) that is commonly given to support recovery from chemotherapy, seems to antagonize CXCR4 effects too, and is claimed to improve AML outcomes by means of this mechanisms, too (105). ILs are an integral component for the regulation of the immune system. AML blasts can secrete a variety of ILs to adjust the TME to their needs: secretion of IL1b promotes growth of blasts, thus, serving as an autocrine growth factor, while suppressing non-neoplastic HSC (106) also by overturning adrenergic signaling in the perivascular niches (see above). By binding circulating IL2 via CD25, AML blasts can reduce T-cell activity (107). IL8 seems to be important for chemoresistance (108), and its secretion by endothelial cells is propagated by AML blasts. IL10 is secreted by MSC of the TME and correlated with worse survival in AML (109). By secreting IL10, MSC can induce immune tolerance, a well-established function of IL10 (110), and thereby shield AML blasts from detection by the immune system.

CONCLUSIONS
In this review, we tried to give an overview of the massively expanding knowledge on the complex interactions of AML with their TME. The delicate structures and functions of the bone marrow niches are profoundly altered in order to help AML blasts to survive, expand and escape immune surveillance. Both, various cellular subsets as well as soluble factors play important roles in this settings ( Figure 6). With more and more knowledge generated from treating solid tumors with immunotherapy, it is now also beginning to be explored in AML, especially in instances treated by chemotherapy. An area still not profoundly investigated is AML in the setting of AHCT. Indeed and as may be anticipated from the information on the interaction of AML with its TME reviewed here, increased knowledge on different pathways and FIGURE 6 | Schematic summarizing some interactions of AML with the TME. Stimulatory signaling is delineated in red, inhibitoryin green; tumor-promoting cells are blueish, tumor-suppressing cellsgreenish, while niche-cells and nutrient-supplying cells are yellowish. The scheme does not claim to be complete and mainly reflects aspects that have been addressed in this review. For abbreviations, we kindly refer to the manuscript body.

Menter and Tzankov
Tumor Microenvironment in AML Frontiers in Immunology | www.frontiersin.org February 2022 | Volume 13 | Article 811144 mechanisms of action mediated through the TMA that may -if therapeutically adjusted -counterbalance graft-versus-host-, while supporting graft-versus-leukemia effects in the setting of AML treated by AHCT has the potential to considerably improve outcome. Further understanding and finding modes of action of immunotherapies and therapies targeting non-immunologic AML-TME interactions to counterbalance the manipulative effects of AML blasts on the bone marrow niche and its constituents has the potential to improve the prognosis of AML patients both at initial stages as well as in the relapse setting and achieving control in the minimal residual disease setting.


s2fieldsofstudy: Biology Medicine

text:
Methylation‐ and homologous recombination deficiency‐related mutant genes predict the prognosis of lung adenocarcinoma

Abstract Background Lung adenocarcinoma (LUAD) is a lung cancer subtype with poor prognosis. We investigated the prognostic value of methylation‐ and homologous recombination deficiency (HRD)‐associated gene signatures in LUAD. Methods Data on RNA sequencing, somatic mutations, and methylation were obtained from TCGA database. HRD scores were used to stratify patients with LUAD into high and low HRD groups and identify differentially mutated and expressed genes (DMEGs). Pearson correlation analysis between DMEGs and methylation yielded methylation‐associated DMEGs. Cox regression analysis was used to construct a prognostic model, and the distribution of clinical features in the high‐ and low‐risk groups was compared. Results Patients with different HRD scores showed different DNA mutation patterns. There were 272 differentially mutated genes and 6294 differentially expressed genes. Fifty‐seven DMEGs were obtained; the top 10 upregulated genes were COL11A1, EXO1, ASPM, COL12A1, COL2A1, COL3A1, COL5A2, DIAPH3, CAD, and SLC25A13, while the top 10 downregulated genes were C7, ERN2, DLC1, SCN7A, SMARCA2, CARD11, LAMA2, ITIH5, FRY, and EPHB6. Forty‐two DMEGs were negatively correlated with 259 methylation sites. Gene ontology and pathway enrichment analysis of the DMEGs revealed enrichment of loci involved in extracellular matrix‐related remodeling and signaling. Six out of the 42 methylation‐associated DMEGs were significantly associated with LUAD prognosis and included in the prognostic model. The model effectively stratified high‐ and low‐risk patients, with the high‐risk group having more patients with advanced stage disease. Conclusion We developed a novel prognostic model for LUAD based on methylation and HRD. Methylation‐associated DMEGs may function as biomarkers and therapeutic targets for LUAD. Further studies are needed to elucidate their roles in LUAD carcinogenesis.

| INTRODUC TI ON
Lung cancer is one of the most common cancers worldwide and has one of the highest mortality rates. 1 Lung adenocarcinoma (LUAD) is a major subtype of lung cancer, accounting for 40% of lung cancers and has an overall 5-year survival rate of less than 20%. [2][3][4] Although the clinical outcomes of patients with LUAD have improved, treatment failure is common and occurs as a result of unresponsiveness or resistance to treatment, tumor recurrence, and metastasis.
Cancers, including LUAD, have high heterogeneity, which is a critical determinant of treatment success and outcomes. Molecular classification has shown a great potential in both distinguishing internal heterogeneity and stratifying LUAD into different subtypes. [4][5][6][7] Several studies have shown that some molecular classifications can predict the prognosis of LUAD. 4,[8][9][10][11] Homologous recombination repair is a conserved process which ensures that DNA is replicated correctly to avoid detrimental mutation-induced damage. 12 However, homologous recombination deficiency (HRD) is common in cancers, and this deficiency leads to impaired DNA repair and drives malignancy. 13,14 Recent studies have reported HRD in lung cancers 15,16 ; Diossy et al. identified a subset of LUAD with HRD, without the loss of the key homologous recombination genes BRCA1/2. 15 Some cancers with HRD showed enhanced responses to poly (ADP-ribose) polymerase inhibitors and platinumbased chemotherapies; therefore, HRD may serve as a biomarker for the response to these drugs. [16][17][18] Although HRD showed prognostic value for ovarian cancer, 19 its efficacy in predicting the prognosis of patients with LUAD remains unexplored.
In this study, we aimed to identify novel prognostic biomarkers for LUAD ( Figure 1). We first investigated global HRD in LUAD. HRD-associated differentially mutated genes (DMGs) and differentially expressed genes (DEGs) were identified to screen for HRDassociated differentially mutated and expressed genes (DMEGs) in LUAD. We then performed DNA methylation profiling of LUAD and analyzed the DMEGs that were negatively correlated with DNA methylation. Finally, we examined the prognostic capacity of methylation-associated DMEGs and built a powerful prognostic model for LUAD. Our study provides insights into the mechanisms underlying LUAD, highlighting the potential of methylation-and HRD-related signatures for predicting clinical outcomes that may improve the clinical management of LUAD.

| Data collection and processing
Raw data, including RNA sequencing data, somatic mutations (single- database. 29 A total of 250 LUAD samples with matched RNA sequencing, SNP, and DNA methylation data were used for subsequent analysis. Ensembl_IDs were converted to Symbol_IDs using the gene mapping annotation from the Gencode database. 30 If multiple Ensembl_IDs corresponded to the same Symbol_ID, the average values for those Ensembl_IDs were calculated and assigned to their corresponding Symbol_ID. Standardized DNA methylation data were obtained after filtering, quality control, and BMIQ normalization using the "ChAMP" package (V2.14.1) in R. 31

| Homologous recombination deficiency analysis
HRD was evaluated as previously described. 32 The HRD score was calculated by examining the following: loss of heterozygosity (LOH), telomeric allelic imbalance (NtAI), and large-scale transition (LST) based on SNP data, after which the samples were divided into high (HRD score ≥42) and low (HRD score <42) HRD score groups as previously described. 33 Global mutations in LUAD were evaluated based on SNP and INDEL data using the "maftools" package in R. 34 The mutation frequency in LUAD was calculated, and the top 10 mutations were visualized.

| Analysis of DMGs
Fisher's test was performed using mafCompare function in the "maftools" package to examine the difference in mutation frequency between the high and low HRD groups, with a cutoff of p < 0.05. 34 The top 10 DMGs were then visualized.

| Analysis of the DEGs between the high and low HRD groups
A t-test was performed to examine the differences in mRNA levels between the high and low HRD groups, using a cutoff of p < 0.05, followed by heatmap and volcano plotting of the DEGs using "gg-plot2" (V3.2.1) in R. 35 The overlapping DEGs and DMGs were considered as DMEGs.

K E Y W O R D S
homologous recombination deficiency, lung adenocarcinoma, methylation, prognosis | 3 of 12 NIE Et al.

| Analysis of methylation-associated DMEGs
Methylated genes were obtained by annotating the methylated sites using the FDb. InfiniumMethylation.hg19 package in R. The Pearson correlation coefficient between the mRNA levels of DMEGs and β values of methylation sites was calculated using the "cor" package in R. The cutoff value was set at p < 0.05 and r < −0.15.

| Gene ontology (GO) and pathway enrichment analysis of the methylation-associated DMEGs
GO and pathway enrichment analysis of the methylation-associated DMEGs were performed using an over-representation analysis in gProfileR, with a cutoff value of Benjamini-Hochberg-adjusted p < 0.05. [36][37][38] F I G U R E 1 Workflow for the identification of a methylation-associated differentially mutated and expressed gene (DMEG) signature for lung adenocarcinoma (LUAD)

| Construction of a prognostic risk model
Tumor samples were randomly and equally divided into training and validation sets (125 samples each). Univariate Cox regression analysis was performed to identify the methylation-associated DMEGs related to the overall survival of patients with LUAD. The prognosis coefficients for multiple factors were calculated using multivariate Cox regression analysis. A prognostic model was built, and risk scores were calculated using the following formula: wherein Coef DMEG represents the prognostic coefficient of the DMEGs calculated using multivariate Cox regression analysis, and Exp DEMG represents the expression level of the DMEGs in the training set.

| Validation of the prognostic model
Risk scores were calculated for the training, validation, and total sets, and patients were divided into high-and low-risk groups based on these scores. Kaplan-Meier survival curves were plotted separately for the three datasets to evaluate the efficacy of the prognostic model.

| Independence analysis of clinical features and risk score
Univariate and multivariate Cox regression analyses were performed to examine whether clinical features, such as tumor, node, metastasis (TNM) stage, age, and sex, and risk score were independent prognostic factors for LUAD. A log-rank p < 0.05 was set as the cutoff value.

| Association between clinical features and risk groups
The differences in clinical features (TNM stage, tumor stage, age, and sex) between the high-and low-risk groups were evaluated using the "ggstatsplot" package (V0.5.0) in R to calculate the ratio of each clinical feature in the different risk groups, and the p value was calculated using the chi-square test. 39

| HRD in LUAD
Scores for the HRD subtypes NtAI, LST, and LOH as well as the total HRD score for the LUAD samples were calculated, and the patients were divided into high (HRD ≥42) and low (HRD <42) HRD score groups (Table S1). The details of the HRD patterns in LUAD are shown in Figure 1. Missense mutations were predominant in LUAD, followed by nonsense mutations (Figure 2A). SNPs were the most frequent variant type in LUAD, while INS and DEL were rare ( Figure 2B). For the single-nucleotide variants, cytosine to adenine mutations were significantly the most frequent among the mutations ( Figure 2C). The number of variants in each LUAD sample was calculated, and the median number was 145 ( Figure 2D). The mutation categories are shown in box plots using different colors ( Figure 2E). The top 10 most frequently mutated  Figure 2F. These genes were highly enriched in the high HRD score group ( Figure 2G).

| Classification of the methylation-associated DMEGs between the high and low HRD score groups
A total of 272 DMGs were identified (Table S2), and DMGs with a p value <0.0001 are shown in Figure 3A. Notably, the HRD of all these genes was significantly higher in the high HRD score group than in the low HRD score group ( Figure 3A). Analysis of the distri-

| Construction and evaluation of the prognostic model
Univariate Cox analysis of 42 methylation-associated DMEGs was performed in the training set, and six were found to be significantly correlated with the prognosis of LUAD (Table 1). EXO1, TPTE, and BMS1 were unfavorable factors that were correlated with worse prognosis in patients with LUAD, while DLC1, KAT2B, and LAMA2 were favorable factors. According to their risk scores, patients in the training, validation, and total sets were divided into high-risk (risk score ≥ medianrisk score ) and low-risk (risk score < median risk score ) groups. Kaplan-Meier survival curves were plotted for each set. In the training set, patients with LUAD in the high-risk group showed significantly shorter overall survival than those in the low-risk group ( Figure 5A). The results were consistent in both the validation and total sets ( Figure 5B,C). The results demonstrate that a prognostic model based on methylationassociated DMEGs can effectively assess risk for patients with LUAD and can be used to stratify them into high-and low-risk groups.

| Identification of independent prognostic factors
The independence of various factors (clinical features and risk score) was evaluated via univariate and multivariate Cox regression analyses. Among the tested parameters (TNM stage, risk score, sex, and age), only the risk score was significant in both the univariate and multivariate Cox regression analyses (Table 2 and Figure S1).

| Association between clinical features and risk group
The distribution of clinical features (TNM stage, tumor stage, age, and sex) in the high-and low-risk groups was examined. The high-risk group contained more male patients, while the low-risk group contained more female patients ( Figure 6A). The tumor stage distribution was also significantly different between the high-and low-risk groups. The high-risk group included more patients with advanced LUAD, while the low-risk group was dominated by patients with early-stage disease ( Figure 6B). Furthermore, the pathologic_N values were different between the two risk groups. Approximately 40% of patients in the high-risk group had tumor cells in nearby lymph nodes compared with 30% of patients in the low-risk group ( Figure 6C). Other clinical features did not differ significantly between the high-and low-risk groups ( Figure S2A-C). HRD is a common feature of cancers, 13,14 and comprehensive studies of HRD have shown its potential in predicting cancer prognosis. 15 Although HRD has been previously reported in LUAD, its prognostic value remains to be evaluated. 15,16 Together with methylation profiling, we developed a methylation-and HRD-based molecular signature that showed good separation of high-and low-risk patients. More importantly, the risk score was an independent prognostic factor for LUAD. This prognostic model exhibits such strong prognostic power as it includes more patients with advanced stage disease and with lymph node metastasis in the high-risk group. suppressive activity in LUAD cells, 41,42 Expression of KAT2B is decreased in many cancers, and KAT2B can regulate SHC3, an immunerelated gene that is associated with the prognosis of LUAD. 43,44 EXO1 is a prognostic biomarker for LUAD and is associated with immune cell infiltration. 45 Our study revealed the relationship between HRD and methylation, further validating its prognostic role in LUAD. The role of TPTE in LUAD has not been reported; this study is the first to report its association with LUAD prognosis, with an F I G U R E 4 Gene ontology (GO) and pathway enrichment analysis of the methylation-associated differentially mutated and expressed genes (DMEGs However, the roles of methylation-associated ECM-related genes in LUAD have not yet been reported. Our study is the first to report the methylation of ECM-related genes in LUAD and suggests their  However, there are some limitations to the current study.

| DISCUSS ION
Although we set up an independent validation set for our prognostic model, it was still within the same cohort as the training set.
Therefore, multiple independent cohorts are required to validate our findings. In addition, while the model exhibited powerful prognostic capacity, and the genes used for its construction were associated with LUAD, further biological studies are required to investigate their roles in LUAD progression and verify our prognostic model.
In summary, the present study profiled the HRD-related gene mutations in LUAD and developed a methylation-associated DMEG signature for predicting the overall survival of patients with LUAD.
Our findings provide a better understanding of LUAD and highlight the potential of methylation-and HRD-related signatures for clinical outcome prediction.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
Conception and design of the research: WY; data acquisition: JL; data analysis and interpretation: YC and YS; statistical analysis: SZ; drafting of the manuscript: GN; manuscript revision for important intellectual content: AZ and JK-L. All authors read and approved the final manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are openly available in The Cancer Genome Atlas (TCGA; http://cance rgeno me.nih.gov/) and Gencode (https://www.genco degen es.org/) databases.


Rubicon-deficiency sensitizes mice to mixed lineage kinase domain-like (MLKL)-mediated kidney ischemia-reperfusion injury

The cytosolic protein rubicon (RUBCN) has been implicated in the removal of necrotic debris and autoimmunity. However, the role of RUBCN in models of acute kidney injury (AKI), a condition that typically involves necrotic kidney tubules, was not investigated. Here, we demonstrate that RUBCN-deficient mice are hypersensitive to renal damage induced by ischemia-reperfusion injury (IRI) and cisplatin-induced AKI. Combined deficiency of RUBCN and mixed lineage kinase domain-like (MLKL) partially reversed the sensitivity in the IRI model suggesting that the absence of RUBCN sensitizes to necroptosis in that model. Necroptosis is known to contribute to TNFα-induced severe inflammatory response syndrome (SIRS), but we detected no statistically significant difference in overall survival following injection of TNFα in RUBCN-deficient mice. We additionally generated RUBCN-deficient mice which lack gasdermin D (GSDMD), the terminal mediator of pyroptosis, but no reversal of the AKI phenotype was observed. Finally, and in contrast to the previous understanding of the role of RUBCN, we did not find a significant autoimmune phenotype in RUBCN-deficient mice, but detected chronic kidney injury (CKD) in aged RUBCN-deficient mice of both sexes. In summary, our data indicate that RUBCN-deficient mice are hypersensitive to kidney injury.

INTRODUCTION
Phagocytosis of dead or dying cells was studied in detail over the last decade [1,2]. The protein rubicon (RUBCN) was initially demonstrated as an autophagy regulator associated with the protein Beclin-1 [3,4], and later demonstrated to be involved in endosomal maturation [5,6]. It is also known that RUBCN recruits NADPH oxidases to participate in the defence against bacteria [7], such as Listeria monocytogenes [8]. Recently, RUBCN was demonstrated to be involved in a specific type of cellular uptake of extracellular content referred to as LC3-associated phagocytosis (LAP) [9][10][11][12]. This process is thought to be critically important in the removal of necrotic debris and may be critical for tissues to allow regeneration following acute injury while avoiding autoimmunity [13].
Acute kidney injury (AKI) is associated with the loss of nephrons and therefore may fundamentally contribute to the progression of chronic kidney disease [14][15][16]. As a clinical consequence of progressive nephron loss, end-stage renal disease (ESRD) requires dialysis treatment or renal transplantation. Acute tubular necrosis is a hallmark condition of AKI, and we reasoned that failure to remove necrotic debris following AKI might affect renal injury in murine models of AKI. We and others recently demonstrated that mixed lineage kinase domain-like (MLKL)-mediated necroptosis is involved in acute kidney injury in mouse models [17][18][19][20] and in humans [21]. Likewise, other forms of regulated necrosis, such as ferroptosis [22,23] and gasdermin D (GSDMD)-mediated pyroptosis [24], are potentially involved in AKI.
Here, we demonstrate that RUBCN-deficient mice are hypersensitive to AKI, an effect that is partially reversed on the combined loss of RUBCN and MLKL, but not RUBCN and GSDMD. In addition, we find aged RUBCN-deficient male mice to develop chronic kidney disease and proteinuria. REAGENT

Mice
Mice of the C57BL/6 N strain, further referred to as wild-type mice, were purchased from the company Charles River, Germany. Rubicon KO (Rubcn KO ) mice on a C57Bl/6 background were described previously [10] and were provided by Douglas R. Green. Mlkl KO breeder pairs were described previously [25] and were kindly provided by James M. Murphy and Warren S. Alexander (The Walter and Eliza Hall Institute of Medical Research, Australia). Breeder pairs of Gsdmd KO mice were provided by Feng Shao (National Institute of Biological Sciences, Beijing, China) and were described previously [26]. Double-knockout animals were generated in the animal facility in Dresden by crossing rubicon-deficient mice to MLKLdeficient or GSDMD-deficient mice. The resulting offspring was then paired up and the F2 generation genotyped. Mice double-deficient for both rubion/MLKLor rubicon/GSDMD, respectively, were then set up as homozygotic breeder pairs. All mice have been housed in individually ventilated cages (IVC, Techniplast Deutschland GmbH) under specified pathogen-free conditions (SPF). The mice were weighed and blood samples were taken until the age of 50 weeks. After 1 year the mice were sacrificed for dissection and the organs were removed for subsequent histological analyses.
All work with animals was carried out in accordance with German animal welfare legislation and was notified and approved by the responsible authority, the Landesdirektion Dresden (TVV 57/2017) if not otherwise indicated. As required by German law, all group sizes were pre-calculated with the help of an independent statistician. The calculation basis aimed for detecting a significant difference with an alpha error of 5% and a beta error of 80%.
Long-term assessment of rubicon-deficient mice Blood collections were done via a puncture of the vena facialis or one of its branches. The samples were kept on ice for 20-30 mins and then centrifuged at 2500 × g for 10 min at 4°C. Fifty microliters were diluted in 100 µl 0.9% sodium chloride solution for assessment of serum creatinine and urea levels (determined by the Clinical Chemistry and Laboratory Medicine, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden) or stored for subsequent ELISA assays.
In week 50, the mice were sacrificed and a blood glucose measurement with a blood glucose monitor was performed. Furthermore, for differential blood counts 200 µl of blood were filled into an EDTA-coated tube and sent to the Clinical Chemistry. Finally, organs were collected (kidneys, spleen, pancreas, caecum, liver, heart, lung), fixed in 4% neutralbuffered paraformaldehyde solution and transferred to 70% ethanol 24 h later. The left kidney was halved, one half was shock-frozen in a 0.5 ml tube in liquid nitrogen for western blot analysis, the other half was transferred to freezer cutting medium (tissue TEK) and shock-frozen in liquid nitrogen.

dsDNA-Ig-and ANA-Enzyme-linked Immunosorbent Assay (ELISA)
Both enzyme-linked immunosorbent assays (ELISAs) were performed according to the manufacturer's protocol (Alpha Diagnostics International). The samples were incubated in multiwell-plates coated with dsDNA or ANAs and visualized using an antibody coupled with horseradish peroxidase-conjugated (HRP). The hydrogen peroxide 3,3,5,5-tetramethylbenzidine (TMB) substrate was added and the optic density (OD) at 450 nm was quantified with a microwell reader (Tecan Group Ltd.).

Histological processing
Periodic acid-Schiff reaction (PAS) and tubular damage score evaluation. Histological processing was carried out at the Histology Facility of BIOTEC, Technische Universität Dresden. The PAS staining was carried out using a PAS-staining Kit (MORPHISTO GmbH). For this, the tissue sections were first placed in xylene for 10 min. Subsequently, the sections were placed in fresh xylene for another 10 min. Now the samples were rehydrated in a descending alcohol series (2 × 100%,2 × 96%,70%,40%). The samples were incubated for 1 min each. The sections were then rinsed with distilled water for 1 min. Subsequently, the sections were incubated for 3 min in 1% periodic acid and washed with tap water for 30 sec afterwards. Schiff's reagent was applied for 2.5 min. Now, the samples were transferred to Haematoxylin (sour according to Mayer) for 3 min. Afterwards washed with tap water (3 min) and distilled water for 10 sec. Then they were dehydrated in an ascending ethanol series for 1 min each (70%,3 × 96%, 2 × 100%). Now, they were placed into xylene two times for 2 min and covered in Cytoseal (Thermo Fisher Scientific Inc.).
The imaging and evaluation of the samples were carried out using the inverse microscope Axiovert 200 M (Carl Zeiss Microscopy GmbH) with the Zeiss AxioCam MRc, Color CCD camera (Carl Zeiss Microscopy GmbH) using the ZEN blue software (Carl Zeiss Microscopy GmbH). Histopathological evaluation was performed in a strictly double-blinded manner.
Tubular damage severity was assessed based on typical morphological criteria of acute renal injury. The criteria were loss of brush border,

Bilateral kidney ischemia and reperfusion injury (IRI)
Bilateral kidney ischemia and reperfusion injury (IRI) was performed as described in detail earlier [23] and a detailed protocol is available [27]. Briefly, 8-12-weeks-old male mice were rigorously matched weight. In the experiment, they were handed over by a second person for blinding of the surgeon. After induction of narcosis, they were placed in a supine position and fixed. The abdomen was opened, the caecum and gut were carefully placed to the side. With the use of a surgical microscope (Carl Zeiss, Jena, Germany), sharp forceps were used to pinch retroperitoneal holes directly cranially and caudally in the renal pedicle. Via this access, a 100 g pressure micro serrefine (FST 18055-03) was placed on the pedicle to induce ischemia and a timer was started. The same procedure was repeated on the other side with a maximal tolerance of 60 s between both clampings. After exactly 30 min, the vascular clamps were removed. Reperfusion was determined visually for both sides before the gut was returned into the abdominal cavity. The parietal peritoneum and the cutis, respectively, were closed separately. Isoflurane application was stopped immediately thereafter, and 1 mL of prewarmed PBS was administered intraperitoneally to compensate for any possible dehydration during surgery. 0.1 µg/g buprenorphine-HCl was administered every 8 h for analgesia. After a 48 h observation period, blood was collected by retro-orbital puncture and the mice were sacrificed.

Cisplatin-induced acute kidney injury
The model of acute kidney injury by cisplatin injection was described previously [23]. Briefly, 8-12-weeks-old female mice were rigorously matched for weight, and then received a single dose of 20 mg/kg cisplatin in 0.9% NaCl in a total volume of 400 µl intraperitoneally. The mice were then returned to their cages and monitored frequently. For analysis of kidney injury, mice were sacrificed after 48 h. Blood was taken and the kidneys were collected for further analysis. For survival assessment following cisplatin application, animals were under permanent observation and survival, as defined by the local authorities, was checked every 2-4 h. In detail, we checked for a combined endpoint of either loss of 20% body weight or the inability to maintain a body temperature higher than 28°C or severe movement imbalances or automutilary behavior.

TNFα-induced systemic inflammatory response syndrome
The model of TNFα-induced shock has been described in detail previously [28]. In our experiments, female 8-12-week-old mice were rigorously weight-matched before mice receiving injections of either vehicle or 25 ng murine TNFα in 200 µL PBS via the tail vein. Under blinding to the genotype, animals were under permanent observation and survival, as defined by the local authorities, was checked every 2-4 h. In detail, we checked for a combined endpoint of either loss of 20% body weight or the inability to maintain a body temperature higher than 28°C or severe movement imbalances or automutilary behaviour.

Analysis of serum creatinine and urea
Blood collections were done via a retro-orbital puncture. The samples were rapidly transferred to the Institute for clinical chemistry and laboratory medicine (Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden) for standardized and completely blinded analysis of serum creatinine and urea levels.

Isolation of murine kidney tubules
Isolation of tubules was performed according to a recently published protocol [23]. The kidneys of 8-week-old male mice were collected and transferred to a petri dish with 2 ml PBS. In the dish the kidneys were decapsulated. The kidney was sliced and those were transferred into a tube containing 1 ml of collagenase solution and incubated for 5 min at 37°C and 850 rpm. This was repeated two more times and those supernatants were transferred into a new tube and the tubules were washed twice with ice-cold incubation solution. The sorting solution was added, and the tubules were distributed on a 24-well plate containing 1 ml of DMEM/F12, Thermo Fischer), supplemented with 0.01 mg/ml recombinant human insulin, 5.5 μg/ml human transferrin, 0.005 μg/ml sodium selenite (Na 2 SeO 3 ) (ITS without linoleic acid, Sigma-Aldrich), 50 nM hydrocortisone, 100 U/ml penicillin, and 100 μg/ml streptomycin (Pen/Strep, Thermo Fisher). Per timepoint (0 h, 0.5 h, 1 h, 2 h, 4 h) and per mouse one well was plated. After the respective timepoints 200 µl of cell suspension was collected and stored on ice for LDH-release assay.

Cell lines and cell culture
Murine NIH-3T3 cells were purchased from the American Type Culture Collection. NIH-3T3 cells were cultured in Dulbecco's modified Eagle medium (DMEM, Thermo Fisher) supplemented with 10% (v/v) FBS (Thermo Fisher), 100 U/ml penicillin, and 100 μg/ml streptomycin (Pen/Strep, Thermo Fisher). The cells were cultured in a humidified 5% CO 2 atmosphere.

siRNA-mediated knockdown of RUBCN
NIH-3T3 cells were plated in a petri dish in 15 ml antibiotic-free medium. After 24 h 180 pmol RNAi and 24 μl Lipofectamine TM (Thermo Fisher) were each mixed in 1.5 ml Opti-MEM® I Medium (Thermo Fisher) without serum, combined and incubated at room temperature for 20 min before dropping the mixture on the cells. The following day, cells were harvested, plated into six-well plates and cell death assays were performed as described below. Knockdown efficacy was determined on protein level using Western Blot.

Western blotting
Cells were lysed in ice-cold 50 mM Tris-HCl, pH 7.

Cell death assays
To induce necroptosis in the NIH-3T3 cell line after siRNA-mediated knockdown, cells were seeded in six-well plates and were stimulated with 20 ng/ml human TNFα (Biolegend), and 20 μM zVAD-fmk (Selleckchem). Ten micromolar Necrostatin-1s (Nec-1s, Merck KGaA) served as protection control. After the indicated timepoints, cells were collected and prepared for flow cytometry.

Flow cytometry
Cells were harvested and the pellets were washed twice in PBS and stained with 5 μl of 7-AAD (BD Biosciences) and 5 μl of annexin-V-FITC (BD Biosciences) added to 100 μl annexin-V binding buffer (BD Biosciences). After 15 min, cells were recorded on the LSRII with the FACS Diva 6.1.1 software (BD Biosciences) and subsequently analyzed with the FlowJo v10 software (Tree Star). The flow cytometry procedure was supported by the Flow Cytometry Core Facility of the CMCB Technology Platform at TU Dresden.

Statistical analysis
Statistical analyses were performed with Prism 8 (GraphPad software, San Diego, CA, USA). Two cohort comparisons of discrete values were analyzed by a one-sided Student's t-test. If more than two groups were involved, a one-way ANOVA was performed with correction by Tukey posthoc test. In both such datasets, the bars represent the mean ± standart deviation if not otherwise indicated. For analyses of survival data, a logrank test was performed. Data were considered significant when *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, or ****p ≤ 0.0001.

RUBCN-deficient mice are hypersensitive to kidney ischemiareperfusion injury
It was previously suggested that RUBCN-deficient mice might exhibit glomerular injury after the age of 20 weeks [29]. However, because necrotic debris occurs within hours in acute kidney injury (AKI)-models [30], we hypothetized that RUBCN might also be involved in the control therein. As demonstrated in Fig. 1A, RUBCN-deficient mice showed higher serum levels of the AKI marker creatinine. Along similar lines, we observed higher levels of serum urea (Fig. 1B), indicating AKI-induced uremia. Histologically, kidney cortical sections were stained for periodic acid Schiff (PAS). More tubular necrosis (Fig. 1C) and a higher tubular damage score (Fig. 1D) were detected. We previously developed a model for the assessment of tubular necrosis in freshly isolated kidney tubules [23]. In that assay, spontaneous LDH release remained unchanged in tubules isolated from RUBCN-deficient mice (Fig. S1). Another preclinical model of renal damage is cisplatin-induced AKI (CP-AKI) in which we typically investigated two timepoints in independent experiments of 48 h and 72 h following cisplatin injection. We detected a trend toward higher levels of serum creatinine (Fig. S2A) and serum urea (Fig. S2B) in the RUBCN-deficient mice, but due to high standard deviations, no statistical significance was reached despite 13 vs. 10 mice per group. However, in an entirely separate approach, we investigated the overall survival of Fig. 2 The hypersensitization of RUBCN-deficient mice is partially reversed on a combined RUBCN/MLKL-double-deficient background. Indicated 8-12-weeks-old male mice underwent bilateral kidney IRI as demonstrated in Fig. 1. 48 h following the onset of reperfusion, serum creatinine (A) and serum urea (B) concentrations were measured and mice were sacrificed. Kidneys were PAS-stained and two representative magnifications are demonstrated in C and quantified using the tubular damage score D. Statistical analysis was performed using one-way ANOVA analysis (p-values are indicated for each experiment).
13 wild-type and 24 Rubcn KO mice following cisplatin injection and detected a significantly shorter time to death in the RUBCNdeficient mice (Fig. S2C, p = 0.006). In conclusion, these data indicate that RUBCN-deficient mice are sensitive to at least two different mouse models of acute kidney injury, and that this effect cannot be directly attributed to kidney tubules.
Combined deficiency of RUBCN and MLKL partially overcomes the sensitization of RUBCN-deficient mice in kidney IRI Several signalling pathways of regulated necrosis have been implicated in the pathogenesis of AKI [22]. However, we considered it unlikely that ferroptosis is the driver of the sensitization of Rubcn KO mice in AKI because renal tubules, in which ferroptosis predominates [23], were not hypersensitive. We therefore decided to investigate MLKL-mediated necroptosis and GSDMD-mediated pyroptosis as hypothetical candidate cell death pathways involved. MLKL-deficient mice, in our hands, were protected from kidney IRI [31]. Indeed, combined deficiency of RUBCN and MLKL partially reversed the significant increase in serum levels of creatinine ( Fig. 2A) and urea (Fig. 2B). PAS staining of the kidney cortex is demonstrated in Fig. 2C and blinded assessment revealed less tubular damage in the Rubcn/Mlkl DKO mice compared to Rubcn KO (Fig. 2D). This result suggested a role for necroptosis in the sensitization of RUBCN-deficient mice to AKI.
To assess a potential role of RUBCN in classical necroptosis in general, we pre-treated NIH-3T3 cells with siRNAs against Rubcn for 48 h (Fig. S3A) and stimulated them for an additional 8 h with TNFα and the pan-caspase inhibitor zVAD-fmk to induce necroptosis. As a protection control, we added the specific RIPK1 inhibitor necrostatin-1s (Nec-1s) [32]. As demonstrated in Fig. S3B, RUBCN knockdown did not affect necroptosis in cell culture. To test the potential role of RUBCN in regulating necroptosis in vivo, we intravenously injected female wild-type and Rubcn KO mice with recombinant TNFα to induce the well-established necroptosis model of severe inflammatory response syndrome (SIRS) [33]. As demonstrated in Fig. S4, no statistically significant difference in overall survival was detected in the RUBCN-deficient mice. In conclusion, these data strongly suggest that RUBCN does not directly regulate necroptosis, but that a non-cell-autonomous effect mediated by necroptosis might explain the partial reversal of the AKI-sensitivity of the RUBCN-deficient mice.
To next test a potential role of pyroptosis, we crossed Rubcn KO mice to Gsdmd KO mice (see methods for details). Combined deficiency of RUBCN and GSDMD does not affect the sensitization of RUBCN-deficient mice in kidney IRI as assessed by serum concentrations of creatinine (Fig. S5A) or urea (Fig. S5B) and did not change the morphological appearance of tubular necrosis in histology (Fig. S5C, D). Fig. 3 Male RUBCN-deficient mice develop slight proteinuria and mild chronic kidney disease at 50 weeks of age. Serum levels of creatinine and urea, and the urine protein to creatinine ratio are depicted for female (upper panels) and male mice (lower panels) of indicated genotypes. Note that the axis for the urine protein to creatinine ratio is higher in the male mice which develop significant proteinuria in contrast to the female littermates. Statistical analysis was performed using one-way ANOVA analysis (*p < 0.05, **p < 0.01, ***p < 0.001). Fig. 4 RUBCN-deficient female mice display splenic atrophy but no signs of autoimmunity at 50 weeks of age. A Male and female RUBCNdeficient mice fail to gain weight over their first 50 weeks of life compared to wild-type littermates (also see Fig. S6 for details). B In contrast to male mice, female mice show splenic atrophy at an age of 50 weeks as estimated by splenic weight. C Male and female wild-type mice and RUBCN-deficient littermates produce indistinguishable levels of autoantibodies against double-stranded DNA at 36 weeks and 50 weeks of age. Statistical analysis was performed one-way ANOVA analysis (*p < 0.05, **p < 0.01, ***p < 0.001).
Male RUBCN-deficient mice develop proteinuria and chronic kidney disease at 50 weeks of age while female RUBCNdeficient mice exhibit splenic atrophy The detection of Rubcn KO mice being sensitive to AKI raised the question of the spontaneous development of chronic kidney injury in these strains. To this end, we set up a one year follow up an experiment with at least 10 mice per group of wild-type mice, RUBCN-deficient mice, Rubcn/Mlkl DKO mice, and Rubcn-Gsdmd DKO mice. This approach was independently followed up for both sexes. At 50 weeks of age, mice were sacrificed and serum creatinine, serum urea and urine protein-to-creatinine ratios were determined as demonstrated in Fig. 3. While both male and female RUBCN-deficient mice developed signs of chronic kidney injury (significant increases in serum urea levels), only male mice on a RUBCN-deficient background additionally presented significant proteinuria. This observation correlated with the failure to gain normal body weight in the Rubcn KO and the Rubcn-Gsdmd DKO mice (Fig. S6A). Interestingly, and somewhat phenocopying the partial reversal of the IRI phenotype, the failure to thrive of Rubcn/Mlkl DKO mice did not reach statistical significance (Fig. S6A). Chronic kidney disease on RUBCN-deficient background did not correlate with renal anemia or erythrocytopenia in the assessment of a standard blood count (Fig. S6B). In this blood test, we additionally observed an increased number of lymphocytes, exclusively in the female mice (Fig. S6B), while serum glucose levels remained unchanged (Fig. S6C). The female lymphocytosis, unexpectedly, correlated with splenic atrophy in all RUBCN-deficient conditions (Fig. 4B), while spleen weight was indistinguishable upon all male groups investigated. Finally, and unlike previously demonstrated, we did not detect a statistically significant increase in double-stranded DNA autoantibodies in the RUBCN-deficient male or female mice at 36 or 50 weeks of age (Fig. 4C). These data indicate that the sole explanation of autoimmunity as a conclusive cause of the failure to gain weight in the Rubcn KO mice is not correct. In summary, we identify RUBCN-deficient mice as a potential model for the investigation of chronic kidney injury.

DISCUSSION
We provide evidence for RUBCN-deficient mice to be sensitive to kidney ischemia-reperfusion injury (IRI). We further demonstrate that pyroptosis-deficiency does not reverse this sensitization, while MLKL-deficiency does. As the knockdown of Rubcn does not affect a standard necroptosis assay, we conclude a non-cell-autonomous involvement of MLKL. Indeed, it was recently demonstrated that metabolites released from dying cells function as tissue messengers [34], and similar mechanisms are potentially involved in models of AKI. However, we previously demonstrated that MLKLdeficient mice are partially but significantly protected in the kidney IRI model and discussed a direct effect of necroptosis in kidney tubules [31]. It is beyond this investigation to identify the detailed mechanism of how RUBCN-deficiency triggers MLKL-mediated necroptosis in this setting, but it is important to realize that pyroptosis does not appear to be involved. It was recently demonstrated that deletion of rubicon from one specific compartment, the renal tubular epithelial cells, did not affect the outcome of kidney ischemia-reperfusion injury [35]. Despite different mouse strains used in that study compared to the one presented here, we interpret these data to be in keeping with our fundings.
As clear as the data on AKI in RUBCN-deficient and Rubcn/ Mlkl DKO mice are, several open questions remain unanswered. In contrast to previous models, we did not detect signs for the development of autoimmunity in Rubcn KO mice [29]. While we do see a failure to develop normal body weight in the RUBCNdeficient and the combined RUBCN/GSDMD-deficient male and female mice, we do not see elevated levels of antinuclear antibodies (ANAs) or autoantibodies against double-stranded DNA. Importantly, we detected lower spleen weight in Rubcn KO female mice compared to control female mice, while no differences were observed in the males. Why RUBCN-deficient mice fail to gain normal body weight in comparison to littermates, therefore, remains an open question.
Another limitation of our study is the lack of explanation of the progressing chronic kidney disease in these mice. In particular, we observed higher urea levels in the RUBCN-deficient male and female mice, alongside an increase in urine protein-to-creatinineratio (proteinuria) exclusively in the male mice. We did not observe any typical signs of glomerulonephritis.
In conclusion, we present evidence for Rubcn KO mice to be significantly sensitive to kidney IRI, an effect that is partially reversed on a combined MLKL-deficient background. In addition, we identify the RUBCN-deficient mice as mildly affected by chronic kidney disease over the first year of life, but we did not detect the development of a severe autoimmune phenotype.

DATA AVAILABILITY
All data needed to evaluate the conclusions in the paper are present in the paper and/or the Supplementary Materials.